Peptide antibiotics and peptide intermediates for their prepartion

ABSTRACT

Novel protected cyclopeptide intermediates are prepared from polymyxin B are used to synthesize new peptide antibiotics. Intermediates are readily derivatized and deprotected to provide new families of antibiotics, which have potent anti-bacterial activity against gram-negative bacteria; but also are useful and potent against gram-positive bacteria.

FIELD OF THE INVENTION

This invention relates to methods for preparing novel protected peptide intermediates from polymyxin B or from related lipopeptide antibiotics, which are used to readily prepare new families of antibiotics that have potent activity against gram-negative and gram-positive bacteria.

BACKGROUND OF THE INVENTION

As and for the background for the present invention, the following references were used (throughout this description of the present invention, the numbers of the references will be referred to):

-   1. Evans, M. E., Feola, D. J., Rapp, R. P. 1999 Polymyxin B Sulfate     and Colistin: Old Antibiotics for Emerging Multiresistant     Gram-Negative Bacteria. The Annals of Pharmacotherapy. 33:960-967. -   2. McCallister, S. M., Alpar H. O., Brown, M. R. W. 1999.     Anti-microbial Properties of Liposomal Polymyxin B. Journal of     Anti-microbial Chemotherapy 43:203-210. -   3. Chihara, S., Ito, A., Yahata, M., Tobita, T., Koyama, Y. 1974.     Chemical Synthesis and Characterization of n-Fattyacyl     Mono-Aminoacyl Derivatives of Colistin Nonapeptide. Agr. Biol. Chem.     38:(10), 1767-1777. -   4. Chihara S., Ito, A., Yahata M., Tobita, T., Koyama, Y. 1973.     Chemical Synthesis and Characterization of α-N-Octanoyl and Other     α-N-Acyl Nonapeptide Derivatives. Agr. Biol. Chem. 37: (12),     2709-2717. -   5. Chihara, S., Ito, A., Yahata, M., Tobita, T., Koyama, Y. 1974.     Chemical Synthesis, Isolation and Characterization of n-N-Fattyacyl     Colistin Nonapeptide with Special Reference to the Correlation     Between Antimicrobial Activity and Carbon Number of Fattyacyl     Moiety. Agr. Biol. Chem. 38:(3), 521-529. -   6. Weinstein, J., Afonso, A., Moss, E. J R., Miller, G. H. 1998.     Selective Chemical Modifications of Polymyxin B. Bioorganic &     Medicinal Chemistry Letters 8:3991-3996. -   7. Tsubery, H., Ofek, I., Cohen, S., Fridkin, M. 2001. N-Terminal     Modifications of Polymyxin B Nonapeptide and Their Effect on     Antibacterial Activity. Peptides. 22: 1675-1681. -   8. Mutter, M., and Bellof, D., A New Base-labile Anchoring Group for     Polymer-supported peptide synthesis, Helv. Chim. Acta, 1984, 67,     2009. -   9. Liu, Y.-Z., Ding, S.-H., Chu, J.-Y. and Felix, A. M., 1990, A     Novel Fmoc-based Anchorage for the Synthesis of protected Peptide on     Solid Phase, Int. J. Pept. Protein Res., 35, 95. -   10. Markou, N., Apostolakos, Koumoudiou, C., Anthanasiou, M.,     Koutsoukou, A., Alamanos I., and Gregorakos, L., 2003, Intravenous     colistin in the treatment of sepsis from multresistant Gran-negative     bacilli in critically ill patients, Critical Care, 7, R78-R83. -   11. Duwe, A. K., Rupar, C. A., Horsman, G. B., and Vas, S. I., 1986.     In Vitro Cytotoxicity and Antibiotic Activity of Polymyxin B     Nonapeptide, Antimicrobial Agents and Chemotherapy, 30:340-341. -   12. Kurihara T., Takeda, H., and Ito, H. 1972, Compounds related to     colistin. V. Synthesis and pharmacological activity of colistin     analogs, Yakugaku Zasshi, 92:129-34. -   13. Parker, W. L., Rathnum, M. L. 1975. EM49, A New Peptide     Antibiotic IV. The Structure of EM49. The Journal of Antibiotics.     28:(5), 379-389. -   14. Hausmann, W., et al., 1954, Polymyxin B1. Fractionation,     molecular-weight determination, amino acid and fatty acid     composition, J. Am. Chem. Soc. 76, 4892-4896. -   15. DeVisser Kriek, N. M. A. J., van Hooft, P. A. V., Van Schepdael     A., Fillipov, D. V., van der Marel, G. A., Overcleeft, H. S., van     Boom, J. H., and Noort, D.m 2003, Synthesis of polymyxin B and     analogues, J. Peptide Res. 61, 298-306. -   16. Kimura, Y., Matsunaga, and Vaara, M., 1992, Polymyxin B     Octapeptide and Polymyxin B Heptapeptide are Potent Outer Membrane     Permeability-Increasing Agents. J. Antibiotics, 45m, 742-749. -   17. Boeck L. D., Fukuda, D. S., Abbott, B. J., Debono, M. 1988.     Deacylation of Actinoplanes utahensis. Journal of Antibiotics     41:(8), 1085-1092. -   18. Kreuzman, A. J., Hodges, R. L., Swartling, J. R., Ghag, S. K.,     Baker, P. J., McGilvray, D., Yeh, W. K. 2000. Membrane-Associated     Echinocandin B Deacylase of Actinoplanes utahensis: Purification,     Characterization, Heterologous Cloning and Enzymatic Deacylation     Reaction. Journal of Industrial Microbiology 24: 173-180. -   19. Boeck, L. D., Fukuda, D. S., Abbott, B. J., Debono, M. 1989.     Deacylation of Echinocandin B by Actinoplanes utahensis. Journal of     Antibiotics 42:(3), 382-388. -   20. Borders, D. B., Curran, W. V., Fantini, A. A., Francis, N. D.,     Jarolmen, H., and Leese, R. A., 2003 Derivatives of Laspartomycin     and Preparation and Use Thereof, U.S. Pat. No. 6,511,962.

Gram-negative bacteria that are resistant to the amino-glycosides, β-lactams, and fluoroquinolones are becoming more common. These bacteria are often susceptible to the polymyxins (Refs. 1, 10). Polymyxin B and the related colistin (polymyxin E) have been used in humans but their use has been previously restricted because of toxicity and the availability of the other less toxic and previously effective antibiotics cited above (Ref. 1). Polymyxin B and colistin are usually administered by intravenous or intramuscular dosing. Colistimethate sodium is a water soluble salt of colistin/formaldehyde/bisulfite and has been of particular therapeutic value in acute and chronic urinary tract infections caused by strains of Pseudomonas aeruginosa (PDR Generics). Polymyxin B sulfate is the drug of choice in the treatment of infections of the urinary tract, meninges, and bloodstream caused by susceptible strains of P. aeruginosa (PDR Generics). Aerosolized polymyxin B is an important component of therapeutic regimens used in the management of pseudomonal lung infections, characteristically found in cystic fibrosis (Ref. 2). Some renal toxicity was observed with recommended dosing of polymyxin B in patients. Neurotoxicity is seen most often in patients with compromised renal function, with an overall incidence of 7.3% reported in one large study with colistin (Ref. 1). When the acyl side chain along with the adjacent diaminobutric acid residue are removed from polymyxin B by an enzyme, the nonapeptide is obtained. The antibacterial activity of this compound is about 2-64 times less potent and the toxicity in cell culture is reduced by about 100 fold (Ref. 11). The in vivo toxicity of the nonapeptide of polymyxin B is significantly less than polymyxin B itself (16). A need for a better version of polymyxin is apparent since it is acceptable for limited use in humans at this time and it is not cross resistant with the aminoglycosides, β-lactams, and fluoroquinolones.

There have been many studies to chemically modify polymyxin and colistin to obtain antibiotics with improved biological properties. Most of these studies were done before good characterization techniques such as HPLC, FABMS and ESIMS were available. In these studies, the nonapeptide has been derivatized by procedures with only some selectivity (Refs. 3, 4, 5, 10, 11, 12). Chromatographic purification methods were limited and many of the products seemed to be relatively impure. More recently, selective chemical modifications of polymyxin B were reported; however, no attempt was made to modify the acyl side chain and amino acids of the side chain (Ref. 6). Chemical selectivity among the basic amino groups of polymyxin B was obtained by pH control. The total synthesis of polymyxin B and four analogs was accomplished by a combination of solid phase peptide syntheses to obtain linear structures followed by removal from the resin and condensation in solution at high dilution to obtain the cyclic peptides (Ref. 7). The derivatives were less active than polymyxin B and one compound contained an Fmoc group in the terminal acyl position. A more recent total synthesis of polymyxin B and a few closely related compounds was accomplished only by solid phase peptide synthesis (Ref. 15). Both of these solid phase total synthetic approaches can provide new derivatives of polymyxin but these methods appear limited compared to the methods of the present invention. The quantities of antibiotic produced are small and require large amounts of amino acid precursors. Any scale up of these methods for clinical studies would be very difficult. The methods described below provide a much better, faster, and efficient synthesis. In addition, the new compounds obtained in our studies have a greater structure diversity with a variety of linkers between the side chain and the cyclic peptide.

OBJECTS AND SUMMARY OF THE INVENTION

Accordingly, a primary object of the present invention is to provide potent and effective antibiotics for treating gram-negative bacteria, without limiting use because of toxicity.

A further object of the present invention is to provide intermediates from polymyxin B or related lipopeptide antibiotics which can be used to prepare new families of antibiotics having potent activity against gram-negative and gram-positive bacteria.

These and other objects of the present invention are provided by protecting the five basic groups of polymyxin B with a sulfonic acid derivative of 9-fluorenylmethoxycarbonyl. This protected form of polymyxin B is polyanionic, water soluble as a salt, and readily reacts in aqueous solution with a particular deacylase enzyme produced by Actinoplanes utahensis. It has not been found to react with polymyxin deacylase or the other enzymes commonly used to produce the nonapeptides of polymyxin or colistin (Ref. 11). The deacylated protected polymyxin peptide, designated the protected PBpeptide-3, has three amino acids in the side chain. By a sequence of modified Edman degradations or enzymatic reactions, it is possible to obtain the protected forms of other new intermediates designated either the protected PBpeptide, PBpeptide-1 or PBpeptide-2 containing zero, one or two amino acids, respectively, in the side chain.

Other acidic protecting groups in other salt forms, which would provide water solubility for the deacylase reaction are possible. Examples of these protecting groups are the carboxylic acid derivatives (Refs. 12, 13) of FMOC. If standard protecting groups such as the FMOC are used, the products have no significant water solubility and an enzyme system in an organic solvent or mixed organic solvent would be required.

The enzyme from Actinoplanes utahensis can be used as the whole broth from the fermentation, the washed cells, or a water-solubilized preparation. The water-solubilized enzyme preparation was obtained by a basic extraction of the washed cells and then the clear extract was adjusted to pH 7-8. This enzyme preparation is the easiest to use, can be freeze-dried to a powder form, and is the most efficient.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

This invention includes methods for preparing four novel protected peptide intermediates from polymyxin B or related lipopeptide antibiotics that are used to prepare new families of anti-biotics which have potent activity against gram-negative, and in some cases gram-positive bacteria. Polymyxin B can be isolated from the fermentation of Bacillus polymyxa according to procedures described in Ref. 14. New antibiotics can be prepared from not only polymyxin B but also from related antibiotics such as other polymyxins, colistin, circulin, and octapeptin (EM49, Ref. 13) by these procedures. The five basic groups of polymyxin B are protected with a sulfonic acid derivative of 9-fluorenylmethoxycarbonyl, as shown in the following structures of the protecting groups:

EXAMPLES OF PROTECTING GROUPS

PROTECTED PB PEPTIDE INTERMEDIATES

protected PBpeptide: R = H

No reaction has been found with polymyxin deacylase or the other enzymes commonly used to produce the nonapeptide of polymyxin or colistin (Refs. 10, 11). The deacylated protected polymyxin peptide, designated the protected PBpeptide-3, has three amino acids in the side chain. By a sequence of modified Edman degradations or enzymatic reactions, it is possible to obtain the protected forms of other new intermediates designated either the protected PBpeptide, PBpeptide-1 or PBpeptide-2 containing zero, one or two amino acids, respectively, in the side chain, shown above.

Other acidic protecting groups in their salt forms which would provide water solubility for the deacylase reaction are possible. Examples of these protective groups are the carboxylic acid derivatives (Refs. 8, 9) of FMOC as shown above. If standard protective groups such as the FMOC are used, the products have no significant water solubility and an enzyme system in an organic solvent or mixed organic solvent would be required. The enzyme from the Actinoplanes utahensis can be used as the whole broth from the fermentation, the washed cells, or a water-solubilized preparation. The water-solubilized enzyme preparation is obtained by a basic extraction of the washed cells and then returning the clear extract to pH 7-8. This enzyme preparation is the easiest to use, can be freeze-dried to a powder form, and is the most efficient.

The enzyme preparation that deacylates the protected polymyxin B can be obtained as a water soluble freeze-dried powder which is relatively stable. The preparation of this enzyme is easily accomplished and requires a unique process involving fermentation of Actinoplanes utahensis, separating the cells from the fermentation, washing the cells with water, extracting the cells with a basic buffer at pH 10 for about 20 minutes, adjusting the extract to pH 7-8 and freeze-drying. The powdered form of the enzyme resulting from this process is relatively stable and can be readily re-dissolved in water for use. Further purification can be obtained by gel filtration chromatography. This enzyme readily deacylates the N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)]₅polymyxin B to obtain the protected polymyxin B peptide. This soluble enzyme was also effective in current studies for deacylation of N-fluorenlymethoxycarbonyl) amphomycin, laspartomycin, and N-fluorenlymethoxycarbonyl)-A21978C which are not polymyxin. The enzyme from Actinoplanes utahensis for deacylation of echinocandin, an antifungal antibiotic, was obtained in a water soluble form by subjecting the cells of A. utahensis to a high concentration of potassium chloride (Refs. 18, 19). This same soluble enzyme preparation was used to deacylate daptomycin which was used as a control (Ref. 18). The (tert-BOC)-A21978C was deacylated with whole broth of Actinoplanes utahensis (Ref. 17). Laspartomycin was also deacylated with whole broth of Actinoplanes utanhesis (Ref. 20).

For the polymyxin-type antibiotics such as the polymyxins, octapeptins, colistin, or circulins the procedures described above can be used to prepare protected intermediates selected from a group consisting of the following or their corresponding salts:

-   -   a) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n)     -   b) H-(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n)     -   c) H-(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n)     -   d) H-peptide-[(2-sulfo)-9-Fmoc]₃     -   wherein for Case 1)         H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n)         -   H is hydrogen, X1 is L-Dab or another amino acid, X2 is             L-Thr or another amino acid, X3 is L-Dab or D-Dab or another             amino acid, and n=3-6;     -   for Case 2) H-(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n)         -   H is hydrogen, X2 is L-Thr or another amino acid, X3 is             L-Dab or D-Dab or another amino acid, and n=3-5;     -   for Case 3) H-(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n)         -   H is hydrogen X3 is L-Dab or D-Dab or another amino acid and             n=3-4; and     -   for Case 4) H-peptide-[(2-sulfo)-9-Fmoc]₃         -   H is hydrogen.

The present invention describes semisynthetic antimicrobial cyclopeptides and their methods of preparation from the PBpeptides or other related peptides derived from collistin, circulin or octapeptin. This invention describes four different PBpeptide intermediates which can be elaborated further to afford the new antimicrobial derivatives, illustrated in the following embodiment:

-   -   1) A-(X1)(X2)(X3)-PBpeptide     -   2) A-(X2)(X3)-PBpeptide     -   3) A-(X3)-PBpeptide     -   4) A-PBpeptide     -   Wherein for Case 1) A-(X1)(X2(X3)-PBpeptide     -   A=R′—(C═O)—, R′—SO₁—, R′—(C═NH)—, R′—NH—(C═S)—, R′—NH—(C═O)—,         R′—CH—, R′—O—(C═O)—, where R′ is alkyl, cycloalkyl, alkenyl,         aryl, heteroaryl, or heterocyclic and X1 is L-Dab or another         amino acid, X2 is L-Thr or another amino acid, and X3 is L-Dab         or another amino acid.     -   For Case 2) A-(X2)(X3)-PBpeptide     -   “A” is the same as described for Case 1, X2 is L-Thr or another         amino acid and X3 is L-Dab or another amino acid.

For Case 3) A-(X3)-BPpeptide

-   -   “A” is the same as described in Case 1 and X3 is L-Dab or         another amino acid.     -   For Case 4) A-PBpeptide, “A” is same as described for Case 1.

Each of these novel PBpeptide intermediates can be used to make new series of potent antibacterial antibiotics by the general procedures described. The desired acids were converted to activated species and then coupled to the protected intermediate PBpeptides and deprotected to give the new antibiotics as represented by compounds 1, 2, 6, 7, 11 and 12 in Table 1. The protected intermediate PBpeptides could also be acylated directly with alkyl or aromatic isocyanates or isothiocyanates to give the corresponding ureas and thioureas and the products deprotected to give other new series of antibiotics as represented by compounds 3-5. The biological activities of some of the new antibiotics prepared from PBpeptides by these procedures are given in Table 1.

Similar to the derivatives of the polymyxin B peptides (PBpeptides), the HSO₃ Fmoc or other acidic Fmoc protected peptides from colistin (Cpeptides), circulin A (CA peptides), octapeptin B (Obpeptin), octapeptin C (OCpeptides), polymyxin A (PApeptides), and polymyxin D (PDpeptides) can be derivatized to give new antibiotics and new antibiotic prodrugs by the described procedures. The structure for these peptides differ from the PBpeptide by the following: Side chain cyclic peptide PBpeptide Dab-Thr-Dab- [Dab-Dab-D-Phe-Leu-Dab-Dab-Thr-] Cpeptide Dab-Thr-Dab- [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr-] CApeptide Dab-Thr- Dab - [Dab-Dab-D-Phe-Ileu-Dab-Dab-Thr-] PApeptide Dab-Thr-D-Dab-  [Dab-Dab-D-Leu-Leu-Dab-Dab-Thr-] PDpeptide Dab-Thr-D-Ser-  [Dab-Dab-D-Leu-Thr-Dab-Dab-Thr-] OBpeptide         D-Dab-  [ Dab-Dab-D-Leu-Leu-Dab-Dab-Leu-] OCpeptide         D-Dab-  [ Dab-Dab-D-Leu-Phe-Dab-Dab-Leu-]

The various new antibiotics described in Table 1 all had good activity against Escherichia coli which is a gram-negative bacterium. The most potent were compounds 1, 4 and 5 which had minimum inhibitory concentrations of the antibiotics at 0.6 micrograms per milliliter. Compounds 2 and 10 were somewhat less potent against Escherichia coli but also had significant activity against Staphylococcus aureus which is a gram-positive bacterium. Therefore some of the new antibiotics can have activity against both gram- and negative and gram-positive bacteria. TABLE 1 Minimum Inhibitory Concentrations (MIC) for New Peptide Antibiotics

Com- E. coli Staph pound R X1 X2 X3 MIC* MIC* Poly- C₈H₁₇CO— Dab Thr Dab 0.6 >10 myxin B 1 n-C₉H₁₉CO— Dab Thr Dab 0.6 2 n-C₁₀H₂₁CO-PAPA-** Dab Thr Dab 1.25 2.5 3 n-C₈H₁₇NHCO— Dab Thr Dab 1.25 10 4 phenyl-NHCS— Dab Thr Dab 0.6 >10 5 phenyl-NHCO— Dab Thr Dab 0.6 >10 6 phenyl-CO— Dab Thr Dab 1.25 >10 7 2-naphthyl-OCH₂—CO— Dab Thr Dab 1.25 >10 8 4-CH₃—C₆H₄—SO₂— Dab Thr Dab 9 n-C₈H₁₇NHCO— — Thr Dab 2.5 >10 10 n-C₁₀H₂₁SO₂— Gly Thr Dab 2.5 5 11 n-C₉H₁₉CO— Lys Thr Dab 2.5 10 12 n-C₉H₁₉CO— Phe Thr Dab *MIC values were determined by serial twofold broth dilution method using Escherichia coli, ATCC #26, and Staphylococcus aureus Smith as assay organisms which were grown in Mueller Hinton broth. **p-aminophenylacetyl

Another variation or series of new antibiotics is represented by the sulfonyl derivatives (Table 1, compounds 8 and 10). The side chains from these compounds are not attached to the PBpeptides by an acyl group but instead a sulfonyl group. Other linkers such as ureas or thioureas have been made and resulted in compounds with good antibacterial activity. Perhaps the most unexpected result was the potent activity of the aromatic acyl side chains and the aromatic groups linked through urea and thiourea linkages (compounds 4 and 5) as shown in Table 1. TABLE 2 New Antibiotics and Intermediates

Compound R X1* X2 X3* P (P)₅Polymyxin B C₈H₁₇CO— Dab-P Thr Dab-P HSO₃-Fmoc- 1 n-C₉H₁₉CO— Dab Thr Dab H 1P n-C₉H₁₉CO— Dab-P Thr Dab-P HSO₃-Fmoc- 2 n-C₁₀H₂₁CO-PAPA-** Dab Thr Dab H 2P n-C₁₀H₂₁CO-PAPA-** Dab-P Thr Dab-P HSO₃-Fmoc- 3 n-C₈H₁₇NHCO— Dab Thr Dab H 3P n-C₈H₁₇NHCO— Dab-P Thr Dab-P HSO₃-Fmoc- 4 phenyl-NHCS— Dab Thr Dab H 4P phenyl-NHCS— Dab-P Thr Dab-P HSO₃-Fmoc- 5 phenyl-NHCO— Dab Thr Dab H 5P phenyl-NHCO— Dab-P Thr Dab-P HSO₃-Fmoc- 6 phenyl-CO— Dab Thr Dab H 6P phenyl-CO— Dab-P Thr Dab-P HSO₃-Fmoc- 7 2-naphthyl-OCH₂—CO— Dab Thr Dab H 7P 2-naphthyl-OCH₂—CO— Dab-P Thr Dab-P HSO₃-Fmoc- 8 4-CH₃—C₆H₄—SO₂— Dab Thr Dab H 8P 4-CH₃—C₆H₄—SO₂— Dab-P Thr Dab-P HSO₃-Fmoc- 9 n-C₈H₁₇NHCO— — Thr Dab H 9P n-C₈H₁₇NHCO— — Thr Dab-P HSO₃-Fmoc- 10 n-C₁₀H₂₁SO₂— Gly Thr Dab H 10P n-C₁₀H₂₁SO₂— Gly Thr Dab-P HSO₃-Fmoc- 11 n-C₉H₁₉CO Lys Thr Dab H 11P n-C₉H₁₉CO Lys-P Thr Dab-P HSO₃-Fmoc- 12 n-C₉H₁₉CO Phe Thr Dab H 12P n-C₉H₁₉CO Phe Thr Dab-P HSO₃-Fmoc- *Dab-P is 4-N—(HSO₃-Fmoc)-diaminobutyryl, Lys-P is 6-N—(HSO₃-Fmoc)-lysyl **PAPA is p-aminophenylacetyl All amino acids are the L-isomers unless indicated otherwise.

The new antibiotics and the new protected antibiotics that were prepared are summarized in Table 2.

Compounds designated 1P, 2P, 3P, 4P, 5P, 6P, 7P, 8P, 9P, 10P, 11P and 12P in Table 2 have the protecting group and would be expected to be pro-drugs. The protected antibiotics according to the present invention are most likely pro-drugs based on work with the corresponding derivatives of insulin and gentamicin. [See Gershonov, E., Goldwaser, I., Fridkin, M., Shechter, Y 2000. A Novel Approach for a Water-Soluble Long-Acting Insulin ProDrug; Design, Preparation, and Analysis of [(2-Sulfo)-9-Fluorenylmethoxycarbonyl]₃-Insulin. Journal of Medicinal Chemistry 43:(13), 2530-2537; and Schechter, Y., Tsubery, H. Fridkin, M. 2002 N-[(2-Sulfo)-9-Fluorenylmethoxycarbonyl]₃-Gentamicin, Is A Long-Acting Prodrug Derivative. Journal of Medicinal Chemistry 45: (19), 4264-4270]. The HSO₃ Fmoc derivatives of insulin, a hormone, and gentamicin, an antibiotic active against gram-negative bacteria, undergo deprotection after they are injected into an animal such as a rat. This liberates the biologically active compound. The new HSO₃Fmoc derivatives of our new antibiotics are listed in Table 2, and would all be expected to be pro-drugs for their corresponding antibiotics. The pro-drug may act as a slow release mechanism for the pharmacokinetics of the antibiotic.

The following Examples are provided to enable a complete understanding of the method of preparation, the intermediates for the preparation of antibiotics and the products obtained from the method, according to the present invention:

Example 1 N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)]₅-polymyxin B

Polymyxin B sulfate (1.0 g., 0.841 mmol.) was dissolved in a solution of 25 ml., saturated sodium bicarbonate, 25 ml. of water and 25 ml. of tetrahydrofuran. A solution of (2-sulfo)-9-fluorenylmethoxy-N-hydroxysuccinimide (2.0 g., 4.8 mmol.) in 25 ml. of tetrahydrofuran was added in several portions over 45 min. The reaction mixture was stirred at room temperature over night and diluted with 50 ml. of water, then acidified with 25 ml of 6N hydrochloric acid to give an oily precipitate. The mixture was chilled and the aqueous layer was decanted and the oily residue was dissolved in 100 ml. of ethanol. The ethanol was evaporated under vacuum (35° C.) and the resulting solid was triturated with ethyl acetate, filtered and dried to afford 1.74 g. of product. HPLC with a gradient on a reverse phase column showed a single peak, N-[2-(sulfo)-9-fluorenlymethoxycarbonyl)]₅-polymyxin B, C₁₃₁H₁₅₀N₁₆O₃₈S₅, when the column eluent was monitered at 215 nm. ESIMS: calc. m/z for C₁₃₁H₁₅₂N₁₆O₃₈S₅, (M+2H)⁺²=1358.4. Found 1358.5

The above procedure can be used with polymyxin free base and 2(sulfo)-9-fluorenylmethoxycarbonyl chloride with similar results.

Example 2 Preparation of the Deacylase

The deacylase is produced by culturing Actinoplanes utahensis NRRL 12052 under submerged aerobic fermentation conditions. Because single-colony isolates from a lyophile of the culture were heterogeneous for both morphology and enzyme production capability, selections were made to recover a stable, high-producing variant. Initially, multiple fermentations were carried out using inocula prepared from strain 12052. Vegetative growth from the flask yielding the best deacylating activity was plated on a differential agar (CM). Colonies were then selected for further evaluation. Generally, small colonies were better enzyme producers than the large colony types. Isolate No. 18 was selected as a small colony type and shown to be the best deacylase producer of all colonies selected. This isolate was routinely used for the production of the deacylase enzyme. CM agar contained corn steep liquor 0.5%, Bacto peptone 0.5%, soluble starch 1.0%, NaCl 0.05%, CaCl₂.2H₂O 0.05% and Bacto agar 2.0%.

The fermentation protocol employed is known (17). A high-producing, natural variant was used in this invention. A stock culture of the NRRL 12052 variant, preserved in 20% glycerol at −70° C., was introduced into a 25×150 mm test tube with a glass rod and Morton closure containing 10 mL of a medium composed of sucrose 2.0%, pre-cooked oatmeal 2.0%, distiller's grains and solubles 0.5%, yeast extract 0.25%, K₂HPO₄ 0.1%, KCl 0.05%, MgSO₄.7H₂O 0.05% and FeSO₄.7H₂O 0.0002% in deionized water. After incubation at 30° C. for 72 hrs on a rotary shaker orbiting at 250 rpm the resulting mycelial suspension was transferred into 50 mL of PM3 medium in a 250-mL Erlenmeyer flask. This medium contained sucrose 2.0%, peanut meal 1.0%, K₂HPO₄ 0.12%, KH₂PO₄ 0.05% and MgSO₄.7H₂O 0.025% in tap water. The flask was incubated at a temperature of 30° C. for a period of 60 to 90 hrs. The harvest time was determined by an assay which involved HPLC analyses of the deacylation of (2-Sulfo-9-fluorenlymethoxycarbonyl)]₅ polymyxin B by the whole broth at different times during the fermentation.

Example 3 Deacylation of the Protected Polymyxin B by the Enzyme in Cells

Precipitation Method: Cells from 450 ml deacylase enzyme were washed 3× with water, then brought back to original volume with 0.02 M ammonium phosphate buffer and adjusted to pH 8.0. N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl)]₅-polymyxin B, 897 mg, was added and the mixture was placed on a shaker at 174 rpm and maintained at 30° C. After five hours the mixture was separated by centrifuging. The clear decant was adjusted to pH 2.3 with 1 N HCl to induce precipitation and allowed to stand at room temperature. The precipitate was separated from the mixture, slurried in 80 ml water, adjusted to pH 6.5 to obtain a clear solution, and freeze-dried to obtain 400 mg of tan powder, the semi-purified salt of the protected peptide, N-[(2-Sulfo)-9-fluorenlymethoxycarbonyl]₅-polymyxin peptide [(NaSO₃-Fmoc)₅-PBP-3]. The cells were extracted with methanol/water to obtain additional material.

Resin Method: Cells from 1 liter deacylase enzyme were washed 3× with water, brought back to original volume with 0.02 M ammonium phosphate buffer, and combined with 2.0 g of N-(2-sulfo)-9-fluorenlymethoxycarbonyl)]₅-polymyxin B. The mixture was placed on a shaker at 175 rpm and maintained at 30° C. for 17 hours. The mixture was then separated by centrifuge and the decant was combined with 20 ml of Amberchrom® CG-161m resin. The resin was washed with 150 ml water, 100 ml 10% CH₃CN:H₂O (3×), and 100 ml 20% CH₃CN:H₂O (2×). The peptide was then eluted 2× with 30% CH₃CN:H₂O, evaporated to remove CH₃CN, and freeze-dried to obtain 283 mg powder, the purified peptide. The remaining peptide was then eluted 3× with 100 ml 50% CH₃CN:H₂O, which were combined evaporated and freeze-dried to obtain 460 mg powder, the purified peptide. The remaining peptide was extracted from the cells 6× with MeOH: H₂O, 100 ml each. The extracts were combined, brought to four liters with water, adjusted to pH 2.1 with sulfuric acid and combined with Amberchrom® CG-161m resin. The resin was rinsed with water and then the peptide was eluted with 100 ml 35% CH₃CN:H₂O, which was evaporated and freeze-dried to obtain 94 mg purified peptide. The remaining peptide was eluted with 50% CH₃CN:H₂O, evaporated to remove CH₃CN, and freeze-dried to obtain 539 mg of the purified peptide. The [(2-Sulfo)-9-fluorenlymethoxycarbonyl)]_(5—)polymyxin B peptide[(HSO₃-Fmoc)₅-PBP-3)] was isolated from these procedures as tan powder, C₁₂₂H₁₃₄N₁₆O₃₇S₅. These materials appeared to be about 75% pure when analyzed by HPLC with the column eluent monitored at 215 nm. ESIMS: calc. m/z for C₁₂₂H₁₃6N₁₆O₃₇S₅: (M+2H)⁺²=1288.4; Found: 1288.

Example 4 Deacylation of the Protected Polymyxin B by the Solubilized Enzyme

Water washed cells from 250 ml of Actinoplanes utahensis fermentation were combined with 125 ml 0.02 M ammonium phosphate buffer, adjusted to pH 10.1 and stirred thirty minutes. The solution of the enzyme was separated by centifuge and adjusted to pH 8.0. This solution could be used directly for deacylations or freeze-dried to obtain a powder form for storage. The decant containing the solubilized enzyme at pH 8.0, was combined with 100 mg (HSO₃-Fmoc)₅-polymyxin B dissolved in 10 ml of CH₃CN:H₂O (1:1), and placed on a shaker at 175 rpm, 84° C. After two hours the completed reaction was removed from the shaker and adjusted to pH 2.0. The precipitate was mixed with 40 ml methanol and the soluble product was separated from a dark precipitate. The solution containing 80 mg of the (HSO₃-Fmoc)₅-PBP-3 was evaporated to 2 ml and added to 10 ml of EtOAc to precipitate the (HSO₃-Fmoc)₅-PBP-3 as a tan powder.

Example 5 Purification of (NaSO₃-Fmoc)₅-PBP-3

About 143 mg of partially purified (NaSO₃-Fmoc)₅-PBP-3 was dissolved in 40 mL of 20% CH₃CN 0.05M in sodium phosphate at pH 6.7. Insolubles were removed by centrifugation. The decant was applied to a styrene-divinylbenzene resin cartridge (Supelco EnviChrom-P®, 25×35 mm) which had been slurry packed and rinsed with 20 mL 20% CH₃CN-0.05M pH 6.7 buffer. Flow rate was about 2 mL/min at RT. The cartridge was eluted with incrementally increasing concentrations of CH₃CN about 0.05M in sodium phosphate at pH 6.7. Collected fractions were evaluated by analytical HPLC. The desired product was eluted with 33% and 40% CH₃CN eluents. Product-containing fractions were pooled and CH₃CN removed under vacuum. The product pool was desalted by adsorption onto a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) which was then rinsed with four 1.0 mL portions of distilled water. Product was stripped from the cartridge with 16 mL of 67% CH₃CN, solvent evaporated under vacuum, pH adjusted to about 5.8 with dilute NaOH, then freeze dried. Yield: 72 mg of a light tan solid, (NaSO₃-Fmoc)₅-PBP-3, C₁₂₂H₁₃₄N₁₆O₃₇S₅. ESIMS: calc. m/z for C₁₂₂H₁₃6N₁₆O₃₇S₅: (M+2H)⁺²=1288.4. Found: 1288.

Example 6 Preparation of N-Phenylthiocarbamyl-(NaSO₃-Fmoc)₅-PBP-3 (Compound 4P)

About 3.9 mg of purified (NaSO₃-Fmoc)₅-PBP-3 was dissolved in 0.20 mL of 75% MeOH 0.25M in pH 8.8 potassium borate, 0.003 mL phenylisothiocyanate was added and stirried at RT. After about 90 min, the reaction mixture was diluted with 4 mL of 0.4M ammonium phosphate at pH 7.2. The product solution was applied to a 0.5 g styrene-divinylbenzene resin cartridge (EnviChrom-P) which was rinsed with 6 mL of 20% CH₃CN 0.10M in sodium phosphate at pH 6.7. Product was eluted using 6 mL of 40% CH₃CN 0.05M in sodium phosphate at pH 6.7. Solvent was evaporated under vacuum and the product was desalted in similar fashion as in Example 5. Yield: 3.5 mg of a white solid, N-phenylthiocarbamyl-(NaSO₃-Fmoc)₅-PBP-3, C₁₂₉H₁₃₇N₁₇O₃₇S₆. ESIMS: calc. m/z for C₁₂₉H₁₃₉N₁₇O₃₇S₆: (M+2H)⁺²=1355.4. Found: 1355.

Example 7 Preparation of N-Phenylthiocarbamyl-PBP-3 (Compound 4)

About 8.4 mg of N-phenylthiocarbamyl-(NaSO₃-Fmoc)₅-PBP-3 was dissolved in 0.20 mL dimethylformamide (DMF), 0.010 mL piperidine was added and stirred at RT for 60 min. The reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.050M acetic acid (pH 5.05) and 0.006 mL acetic acid and 4 mL MeOH. The clear solution was applied to a CM-Sepharose® column (10×20 mm, ca. 2 mL volume) which had been conditioned with 50% MeOH 0.05M in ammonium acetate buffer at pH 5.0. The sample-loaded column was rinsed with 4 mL 50% MeOH-0.05M ammonium acetate pH 5.0 then with 4 mL 0.05M ammonium acetate pH 5.0 buffer. Product was eluted with 8 mL of 0.27 M sodium sulfate at pH 2.3. The product was further purified by application onto a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) which was eluted with incrementally increasing concentrations of CH₃CN 0.05M in pH 2.3 sodium sulfate; product was eluted with 20% CH₃CN. Solvent was removed under vacuum from the product-containing fraction pool which was then desalted in similar fashion as in Example 5, pH adjusted to 6.3 then freeze dried. Yield: 2.7 mg of a white solid, PTC-PBP-3, C₅₄H₈₇N₁₇O₁₂S.

FABMS: calc. for C₅₄H₈₈N₁₇O₁₂S:(M+H)⁺=1198.7. Found: 1198.5(M+H)⁺, 1220.4 (M+Na)⁺.

Example 8 Preparation of (NaSO₃-Fmoc)₄-PBP-2 (protected nonapeptide)

About 2.9 mg N-phenylthiocarbamyl-(NaSO₃-Fmoc)₅-PBP-3 (from Example 6) was dissolved in 0.30 mL of anhydrous trifluoroacetic acid (TFA) and heated in a 50° C. water bath for 15 min. TFA was evaporated with a stream of dry nitrogen and the residue was dissolved in 12 mL 0.20M ammonium phosphate at pH7.2 and 6 mL CH₃CN containing 49 mg triglycine (as an acylating agent scavenger). To remove potentially reactive intermediates, the product solution was first applied to a 0.5 g styrene-divinylbenzene resin cartridge (EnviChrom-P) which was eluted with 10 mL of 40% CH₃CN 0.04M in ammonium phosphate at pH 7.2. Product-containing fractions were pooled (˜18 mL), diluted with 12 mL distilled water, then applied to a fresh 0.5 g resin cartridge which was eluted with incrementally increasing concentrations of CH₃CN about 0.05M in ammonium phosphate at pH 7.2. Product-containing fractions were pooled (˜12 mL), 8 mL of 0.54M sodium sulfate pH 2.3 buffer added, and the product was desalted in similar fashion as in Example 5. The solution pH was adjusted to 5.9 and the sample freeze dried. Yield: 1.8 mg of a white solid, (NaSO₃-Fmoc)₄-PBP-2, C₁₀₃H₁₁₆N₁₄O₃₁S₄ ESIMS: calc. m/z for C₁₀₃H₁₁₈N₁₄O₃₁S₄ (M+2H)⁺²=1087.3. Found: 1087.

Example 9 Preparation of n-Decanoyl-PBP-3 (Compounds 1 and 1P)

About 21.8 mg of partially purified (NaSO₃-FMoc)₅-PBP-3 was dissolved in 0.20 mL DMF, 0.020 mL distilled water, and 0.030 mL saturated NaHCO₃ (pH-8.9); 4.5 mg n-decanoyl-N-hydroxysuccinimide (C₁₀—OSu) was added and stirred at RT for 55 min (about 82% conversion by HPLC). An additional 1.8 mg of C₁₀—OSu was added; after 20 min at RT conversion to Compound 1P was at least 95%. To the reaction mix was added 0.010 mL piperidine. After 35 min at RT the reaction mixture was diluted with 4 mL 0.25M ammonium sulfate at pH2.3 yielding a very milky mixture at pH 3.0 which was extracted with 4 mL ethylacetate; the product-containing aqueous phase was diluted with 4 mL distilled water. Product was initially isolated by size exclusion chromatography on a Sephadex® G-25 column (2.5×40 cm) eluted with 0.10M ammonium sulfate at pH 2.3. Product-containing fractions were pooled and further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) by elution with incrementally increasing concentrations of CH₃CN about 0.05M in sodium phosphate at pH 6.7; product was eluted with 25% CH₃CN. Product was desalted and freeze dried in similar fashion as in Example 5. Yield: 2.5 mg of a white solid, Compound 1, C₅₇H₁₀₀N₁₆O₁₃. FABMS: calc. for C₅₇H₁₀₁N₁₆O₁₃, (M+H)⁺=1217.8. Found: 1217(M+H)⁺, 1239(M+Na)⁺.

Example 10 Preparation of N-(n-Decanoyl)-p-aminophenylacetyl-PBP-3(Compounds 2 and 2P)

About 18.5 mg of purified (NaSO₃-Fmoc)₅-PBP-3 was dissolved in 0.20 mL DMF and 0.030 mL saturated NaHCO₃ (pH˜8.9); 5.8 mg N-(n-decanoyl)-p-aminophenylacetyl-N-hydroxysuccinimide (C₁₀-PAPA-OSu) was added and stirred at RT for 50 min (about 69% conversion by HPLC). An additional 2.5 mg of C₁₀-PAPA-OSu was added; after 30 min at RT conversion to Compound 202P was about 87%. To the reaction mix was added 0.010 mL piperidine. After 25 min at RT the reaction mixture was diluted with 5 mL 0.20M ammonium sulfate at pH2.3 yielding a very milky mixture at pH 2.8 which was extracted with 5 mL ethylacetate. Product was initially isolated by size exclusion chromatography on a Sephadex® G-25 column (2.5×40 cm) eluted with 0.10M ammonium sulfate at pH 2.3. Product-containing fractions were pooled and further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) by elution with incrementally increasing concentrations of CH₃CN about 0.10M in ammonium sulfate at pH 2.3; product was eluted with 30% CH₃CN. Further purification was achieved using size exclusion chromatography on a Sephadex LH-20 column (2.5×40 cm) eluted with 0.026M ammonium acetate/0.053M acetic acid in MeOH. Product-containing fractions were evaporated under vacuum to near dryness, the residue dissolved in 10 mL distilled water, then desalted and freeze dried in similar fashion as in Example 5. Yield: 2.5 mg of a white solid, Compound 2, C₆₅H₁₀₇N₁₇O₁₄. FAMS: calc. for C₆₅H₁₀₈N₁₇O₁₄, (M+H)⁺=1350.8. Found: ¹³⁵I(M+H)⁺, 1373(M+Na)⁺.

Example 11 Preparation of n-Octanoylcarbamyl-PBP-3 (Compounds 3 and 3P)

About 10.2 mg of purified (NaSO₃-Fmoc)₅-PBP-3 was dissolved in 0.20 mL DMF and 0.020 mL distilled water; 0.003 mL octylisocyanate was added and stirred at RT for 60 min (about 31% conversion by HPLC). To the reaction mixture was added 0.020 mL saturated NaHCO₃ pH 8.9; after 20 min at RT conversion to Compound 3P was about 91%. After another 35 min at RT 0.010 mL piperidine was added. After 30 min at RT the reaction mixture was diluted with 4 mL 0.026M ammonium acetate/0.053M acetic acid in MeOH and the product was isolated by size exclusion chromatography on a Sephadex LH-20 column as in Example 10. Product-containing fractions were evaporated under vacuum to near dryness, the residue dissolved in 8 mL distilled water, then desalted and freeze dried in similar fashion as in Example 5. Yield: 2.3 mg of a white solid, Compound 3, C₅₆H₉₉N₁₇O₁₃. FABMS: calc. for C₅₆H₁₀₀N₁₇O₁₃, (M+H)⁺=1218.8. Found: 1219(M+H)⁺, 1241(M+Na)⁺.

Example 12 Preparation of Phenylcarbamyl-PBP-3 (Compounds 5 and 5P)

About 19.8 mg of purified (NaSO₃-Fmoc)₅-PBP-3 (ca. 85%) was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO₃ pH 8.7; 0.005 mL phenylisocyanate was added and stirred at RT to obtain Compound 5P. After 45 min 0.020 mL of piperidine was added. After 45 min at RT the reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.05M acetic acid and 0.012 mL acetic acid and 4 mL MeOH yielding a clear solution at apparent pH 6.2. The product was isolated on a CM-Sepharose column as in Example 7. The product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN 0.05M in sodium sulfate at pH 2.3; product was eluted with 20% CH₃CN. Product-containing fractions were desalted and freeze dried in similar fashion as in Example 5. Yield: 4.4 mg of a white solid, Compound 5, C₅₄H₈₇N₁₇O₁₃. FABMS: calc. for C₅₄H₈₈N₁₇O₁₃, (M+H)⁺=1182.7. Found: 1183(M+H)⁺, 1205(M+Na)⁺, 1221(M+K)⁺.

Example 13 Preparation of Benzoyl-PBP-3 (Compounds 6 and 6P)

About 20.4 mg of purified (NaSO₃-Fmoc)₅-PBP-3 (ca. 85%) was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO₃ pH 8.0; 8.4 mg of benzoyl-N-hydroxysuccinimide was added and stirred at 29° C. to obtain Compound 6P. After 60 min 0.020 mL of piperidine was added. After 20 min at RT the reaction mixture was diluted with 4 mL 0.10M ammonium acetate-0.05M acetic acid and 0.013 mL acetic acid and 4 mL MeOH yielding a clear solution. The product was isolated on a CM-Sepharose column as in Example 7. The product was further purified by preparative HPLC using a Delta-Pak® C18 column (25×210 mm, Waters Corp.) eluted with an isopropanol gradient (18%-23% over 100 min, linear, at 5 mL/min) buffered with 0.05M sodium sulfate at pH2.5. Product-containing fractions were pooled, solvent removed under vacuum, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 1.6 mg of a white solid, Compound 6, C₅₄H₈₆N₁₆O₁₃. FABMS: calc. for C₅₄H₈₇N₁₆O₁₃, (M+H)⁺=1166.7. Found: 1167(M+H)⁺, 1189(M+Na)⁺.

Example 14 Preparation of 2-Naphthoxyacetyl-PBP-3 (Compounds 7 and 7P)

About 19.0 mg of purified (NaSO₃-Fmoc)₅-PBP-3 was dissolved in 0.40 mL of 75% MeOH 0.25M in pH 8.8 potassium borate; 6.3 mg of 2-naphthoxyacetyl-N-hydroxysuccinimide was added and stirred at RT to produce Compound 7P. After 35 min 0.020 mL piperidine was added. After 30 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% CH₃CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 4.7 mg of a white solid, Compound 7, C₅₉H₉₀N₁₆O₁₄.

FABMS: calc. for C₅₉H₉₁N₁₆O₄, (M+H)⁺=1247.7. Found: 1247(M+H)⁺, 1269(M+Na)⁺.

Example 15 Preparation of 4-Methylphenylsulfonyl-PMB-3 (Compounds 8 and 8P)

About 21.7 mg of purified (NaSO₃-Fmoc)₅-PBP-3 was dissolved in 0.50 mL of 75% MeOH 0.25M in pH 8.8 potassium borate; 5.1 mg of 4-methylphenylsulfonyl chloride (tosyl chloride) was added and stirred at RT to produce Compound 8P. After 60 min 0.020 mL piperidine was added. After 30 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 20% CH₃CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 4.9 mg of a white solid, Compound 8, C₅₄H₈₈N₁₆O₁₄S. FABMS: calc. for C₅₄H₈₉N₁₆O₁₄S (M+H)⁺=1217.6, Found: 1217(M+H)⁺, 1239(M+Na)⁺.

Example 16 Preparation of n-Octanoylcarbamyl-PBP-2 (Compounds 9 and 9P)

About 10.1 mg of purified (NaSO₃-Fmoc)₄-PBP-2 was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO₃ pH 8.7; 0.003 mL octylisocyanate was added and stirred at RT to produce Compound 9P. After 45 min at RT 0.020 mL piperidine was added. After 40 min at RT the reaction mixture was diluted with 10 mL 20% CH₃CN containing 0.014 mL H₂SO₄. Product was isolated on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% and 30% CH₃CN. Product-containing fractions were pooled, solvent removed under vacuum, 2 mL 1.0M ammonium acetate pH 5.0 buffer was added, pH adjusted to 5.0 by addition of ca. 0.6 mL 1.5M NH₄OH, and then diluted with an equal volume of MeOH. Product was isolated via CM-Sepharose chromatography as in Example 7. The product was further purified on a 0.5 g styrene-divinylbenzene cartridge as above but using eluents at pH 9.9 (0.10M NH₄OH-0.01M (NH₄)₂SO₄); product eluted with 30% CH₃CN. Product-containing fractions were pooled, solvent removed under vacuum, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 2.7 mg of a white solid, C₅₂H₉₂N₁₆O₁₁. FABMS: calc. for C₅₂H₉₃N₁₆O₁₁, (M+H)⁺=1118.7, Found: 1119(M+H)⁺, 1141(M+Na)⁺, 1157(M+K)⁺.

Example 17 Preparation of N-(n-Decylsulfonyl)glycyl-PBP-2 (Compounds 10 and 10P)

About 9.7 mg of purified (NaSO₃-Fmoc)₄-PBP-2 was dissolved in 0.20 mL DMF and 0.020 mL saturated NaHCO₃ pH 8.7; 5.2 mg of n-decylsulfonamidoglycyl-N-hydroxy succinimide was added and stirred at RT to produce Compound 10P. After 45 min 0.020 mL piperidine was added. After another 40 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 33% CH₃CN. Product-containing fractions were pooled, solvent removed under vacuum, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 1.5 mg of a white solid, Compound 10, C₅₅H₉₇N₁₅O₁₄S. FABMS: calc. for C₅₅H₉₈N₁₅O₁₄S, (M+H)⁺=1224.7. Found: 1224.5(M+H)⁺, 1246.5(M+Na)⁺.

Example 18 Preparation of 2-N-(n-Decanoyl)lysyl-PMB-2 (Compounds 11 and 11P)

About 10.3 mg of purified (NaSO₃-Fmoc)₄-PBP-2 was dissolved in 0.20 mL of DMF and 0.020 mL of saturated NaHCO₃ pH8.3; 8.7 mg of 6-N-Fmoc-2-N-(n-decanoyl)-lysyl-N-hydroxysuccinimide was added and stirred at RT to produce Compound 11P. After 50 min 0.020 mL piperidine was added. After 45 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 25% CH₃CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 2.0 mg of a white solid, Compound 11, C₅₉H₁₀₄N₁₆O₁₃. FABMS: calc. for C₅₉H₁₀₅N₁₆O₁₃, (M+H)⁺=1245.8, Found: 1245(M+H)⁺, 1267(M+Na)⁺.

Example 19 Preparation of N-(n-Decanoyl)phenylalanyl-PMB-2 (Compounds 12 and 12P)

About 10 mg of purified (NaSO₃-Fmoc)₄-PBP-2 was dissolved in 0.40 mL of 75% MeOH 0.25M in pH 8.8 potassium borate; 6 mg of N-(n-decanoyl)-phenylalanyl-N-hydroxysuccinimide was added and stirred at RT to produce Compound 12P. After 50 min 0.020 mL piperidine was added. After 45 min at RT the reaction mixture was diluted and product isolated on a CM-Sepharose column as in Example 7. Product was further purified on a 0.5 g styrene-divinylbenzene cartridge (EnviChrom-P) using incrementally increasing concentrations of CH₃CN about 0.05M in sodium sulfate at pH2.3; product was eluted with 33% CH₃CN. Product-containing fractions were pooled, diluted with an equal volume of distilled water, and product was desalted and freeze dried in similar fashion as in Example 5. Yield: 3.0 mg of a white solid, Compound 12, C₆₂H₁₀₁N₁₅O₁₃. FABMS: calc. for C₆₂H₁₀₂N₁₅O₁₃ (M+H)⁺=1264.8. Found: 1265(M+H)⁺, 1287(M+Na)⁺.

Example 20 Preparation of (NaSO₃-Fmoc)₄-PBP-1 (protected octapeptide)

The procedure used in Example 8 to prepare the (NaSO₃-Fmoc)₄-PBP-2 from (NaSO₃-Fmoc)₅-PBP-3 was repeated starting with (NaSO₃-Fmoc)₄-PBP-2, 50 mg, to remove the threonine residue and yielding (NaSO₃-Fmoc)₄-PBP-1, 37 mg, as a white powder, with the expected HPLC relative retention time.

Example 21 Preparation of (NaSO₃-Fmoc)₃-PBP (protected heptapeptide)

(NaSO₃-Fmoc)₄-PBP-2 (5.0 mg) was dissolved in 1.4 mL of 0.02M sodium citrate buffer (pH6.4) and 0.006M in 2-mercaptoethylamine(MEA) hydrochloride. The enzyme solution was prepared by dissolving 10 units (0.9 mg) of cathepsin C (dipeptidyl aminopeptidase) (EC 3.4.14.1) in 11.0 mL of the citrate-MEA buffer. Enzyme solution (0.25 mL) was added to the peptide solution which was incubated at about 37° C. for about 24 hours. HPLC analysis indicated about 75% conversion to (NaSO₃-Fmoc)₃-PBP. The incubated solution was diluted with 11.0 mL 0.4M pH7.2 ammonium phosphate buffer, 3.0 mL distilled water and 0.5 mL CH₃CN. The product was isolated on an ENVI-Chrom-P 0.5 g resin cartridge; product was eluted with 25% CH₃CN, 0.05M in pH7.2 buffer. After HPLC evaluation, product-containing fractions (accounting for about 72% of the total product) were pooled, desalted and freeze dried as in Example 8. Yield: 1.0 mg of a pale yellow solid, 87% by HPLC (215 nm area %), with the expected HPLC relative retention time for the (NaSO₃-Fmoc)₃-PBP.

While the foregoing description of the preferred and and alternative embodiments of the present invention is considered sufficient, the only limitations thereof are in the following claims: 

1. A method for preparing an intermediate for use in the synthesis of a new peptide antibiotic, including the steps of: (a) Protecting the amino groups of the polymyxins or other related antibiotics chosen from the group consisting of colistin, a circulin, and an octapeptin, with (2-sulfo)-9-fluorenylmethoxycarbonyl or another acidic derivative of 9-fluorenylmethoxycarbonyl; (b) Treating the product from the reaction of step (a) with a deacylase to provide a protected peptide intermediate; and (c) Using a modified Edman degradation method or peptidase enzymatic reaction to obtain another protected intermediate peptide by reducing in size by one to three amino acids in the exocyclic peptide side chain of the protected peptide.
 2. A method for producing an antibiotic active against gram-negative and gram-positive bacteria, including strains resistant to clinically used antibiotics, comprising the steps of: (a) Protecting the amino groups of the polymyxins or other related antibiotics chosen from the group consisting of colistin, a circulin, and an octapeptin, with (2-sulfo)-9-fluorenylmethoxycarbonyl or another acidic derivative of 9-fluorenylmethoxycarbonyl; (b) Treating the product from the reaction of step (a) with a deacylase to provide a protected peptide intermediate; (c) Using a modified Edman degradation method or peptidase enzymatic reaction to obtain another protected intermediate peptide by reducing in size by one to three amino acids in the exocyclic peptide side chain of the protected peptide. (d) Chemically modifying the intermediate to produce a protected antibacterial derivative; and (e) Removing the acidic protecting groups to produce the antibiotic.
 3. An intermediate, which is a chemically protected form of a peptide derived from the polymyxins, octapeptins, colistin, or circulins, and selected from a group consisting of the following, or their corresponding salts: Case 1) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n) Case 2) H-(X2) (X3)-peptide-[(2-sulfo)-9-Fmoc]_(n) Case 3) H-(X2)-peptide-[(2-sulfo)-9-Fmoc]_(n) Case 4) H-peptide-[(2-sulfo)-9-Fmoc]₃ wherein for Case 1) H-(X1)(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n) H is hydrogen, X1 is L-Dab or another amino acid, 2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, and n=3-6; for Case 2) H-(X2)(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n) H is hydrogen, X2 is L-Thr or another amino acid, X3 is L-Dab or D-Dab or another amino acid, and n=3-5; for Case 3) H-(X3)-peptide-[(2-sulfo)-9-Fmoc]_(n) H is hydrogen X3 is L-Dab or D-Dab or another amino acid and n=3-4; and for Case 4) H-peptide-[(2-sulfo)-9-Fmoc]₃ H is hydrogen.
 4. Acidic protected peptide intermediates, derived from the corresponding protected polymyxin B, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably HSO₃-Fmoc and the protected peptide intermediates have the structure:

protected PBpeptide: R = H


5. Acidic protected peptide intermediates, derived from the corresponding protected colistin, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably. HSO₃-Fmoc- and the protected peptide intermediates have the structure:

protected Cpeptide: R = H


6. Acidic protected peptide intermediates, derived from the corresponding protected circulin A, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably HSO₃-Fmoc- and the protected intermediates have the structure:

protected CApeptide: R = H


7. Acidic protected peptide intermediates, derived from the corresponding protected octapeptin, which can be used to synthesize new peptide antibiotics or their prodrugs where the protecting group is preferably HSO₃-Fmoc- and the protected peptide intermediates have the structure:

P = Protective group protected octapeptin peptide: R = H


8. The protected peptide intermediate of claim 7, wherein another component of the octapeptin antibiotic includes L-phenylalanine instead of L-leucine at the 5-position and wherein the component forms a similar, but alternative, protected peptide intermediate.
 9. An antibacterial compound or protected compound prepared from a chemically protected form of PBpeptide having the following structure where p equals the protective group HSO₃-Fmoc- or hydrogen:

Compound R X1* X2 X3* P (P)₅Polymyxin B C₈H₁₇CO— Dab-P Thr Dab-P HSO₃-Fmoc- 1 n-C₉H₁₉CO— Dab Thr Dab H 1P n-C₉H₁₉CO— Dab-P Thr Dab-P HSO₃-Fmoc- 2 n-C₁₀H₂₁CO-PAPA-** Dab Thr Dab H 2P n-C₁₀H₂₁CO-PAPA-** Dab-P Thr Dab-P HSO₃-Fmoc- 3 n-C₈H₁₇NHCO— Dab Thr Dab H 3P n-C₈H₁₇NHCO— Dab-P Thr Dab-P HSO₃-Fmoc- 4 phenyl-NHCS— Dab Thr Dab H 4P phenyl-NHCS— Dab-P Thr Dab-P HSO₃-Fmoc- 5 phenyl-NHCO— Dab Thr Dab H 5P phenyl-NHCO— Dab-P Thr Dab-P HSO₃-Fmoc- 6 phenyl-CO— Dab Thr Dab H 6P phenyl-CO— Dab-P Thr Dab-P HSO₃-Fmoc- 7 2-naphthyl-OCH₂—CO— Dab Thr Dab H 7P 2-naphthyl-OCH₂—CO— Dab-P Thr Dab-P HSO₃-Fmoc- 8 4-CH₃—C₆H₄—SO₂— Dab Thr Dab H 8P 4-CH₃—C₆H₄—SO₂— Dab-P Thr Dab-P HSO₃-Fmoc- 9 n-C₈H₁₇NHCO— — Thr Dab H 9P n-C₈H₁₇NHCO— — Thr Dab-P HSO₃-Fmoc- 10 n-C₁₀H₂₁SO₂— Gly Thr Dab H 10P n-C₁₀H₂₁SO₂— Gly Thr Dab-P HSO₃-Fmoc- 11 n-C₉H₁₉CO Lys Thr Dab H 11P n-C₉H₁₉CO Lys-P Thr Dab-P HSO₃-Fmoc- 12 n-C₉H₁₉CO Phe Thr Dab H 12P n-C₉H₁₉CO Phe Thr Dab-P HSO₃-Fmoc- *Dab-P is 4-N—(HSO₃-Fmoc)-diaminobutyryl, Lys-P is 6-N—(HSO₃-Fmoc)-lysyl **PAPA is p-aminophenylacetyl All amino acids are the L-isomers unless indicated otherwise.


10. Peptide antibiotics having the following structure and concentration for use against gram-positive and gram-negative bacteria:

Com- E. coli Staph pound R X1 X2 X3 MIC* MIC* Poly- C₈H₁₇CO— Dab Thr Dab 0.6 >10 myxin B 1 n-C₉H₁₉CO— Dab Thr Dab 0.6 2 n-C₁₀H₂₁CO-PAPA-** Dab Thr Dab 1.25 2.5 3 n-C₈H₁₇NHCO— Dab Thr Dab 1.25 10 4 phenyl-NHCS— Dab Thr Dab 0.6 >10 5 phenyl-NHCO— Dab Thr Dab 0.6 >10 6 phenyl-CO— Dab Thr Dab 1.25 >10 7 2-naph- Dab Thr Dab 1.25 >10 thyl-OCH₂—CO— 8 4-CH₃—C₆H₄—SO₂— Dab Thr Dab 9 n-C₈H₁₇NHCO— — Thr Dab 2.5 >10 10 n-C₁₀H₂₁SO₂— Gly Thr Dab 2.5 5 11 n-C₉H₁₉CO— Lys Thr Dab 2.5 10 12 n-C₉H₁₉CO— Phe Thr Dab *MIC values were determined by serial twofold broth dilution method using Escherichia coli, ATCC #26, and Staphylococcus aureus Smith as assay organisms which were grown in Mueller Hinton broth. **p-aminophenylacetyl


11. An antibiotic prepared from an intermediate, which is a chemically protected form of a peptide derived from the polymyxins, octapeptins, colistin, or circulins, said antibiotic selected from a group consisting of the following, or their corresponding salts: Case 1) A-(X1)(X2)(X3)-peptide Case 2) A-(X2)(X3)-peptide Case 3) A-(X3)-peptide Case 4) A-peptide wherein for Case 1) A-(X1)(X2)(X3)-peptide A=R′—(C═O)—, R′—SO₂—, R′—(C═NH)—, R′—NH—(C═S)—, R′—NH—(C═O)—, R′—O—(C═O)—, R′CH₂— where R′ is alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, or heterocyclic and X1 is L-Dab or another amino acid, X2 is L-Thr or another amino acid, and X3 is L-Dab, D-Dab or another amino acid, excluding colistin peptides where X1 is an aliphatic amino acid and where R′(C—O)— is alkyl, for Case 2) A-(X2)(X3)-peptide “A” is the same as described for Case 1, X2 is L-Thr or another amino acid and X3 is L-Dab, D-Dab or another amino acid, excluding N-acyl colistin nonapeptide derivatives where R′ is alkyl, aryl, or cycloalkyl, for Case 3) A-(X3)-peptide “A” is the same as described in Case 1, and X3 is L-Dab, D-Dab or another amino acid, excluding R′—(CO)— where R′ is alkyl for octapeptin peptides, for Case 4) A-peptide “A” is the same as described for Case
 1. 